Before hybridization, labeled aRNA samples were fragmented (Fragmentation Reagent Kit, Ambion) according to manufacturer's instructions.
The labeled aRNA was purified using an AminoAllyl MessageAmp™ II aRNA Amplification Kit (Ambion) according to the manufacturer's instructions and fragmented using fragmentation reagents (Ambion).
Isolated RNA was enriched for mRNA by using oligo dT Dynabeads (Invitrogen) according to the manufacturer's instructions and fragmented into fragments of 200 700 nucleotides by incubating at 70°C for 15 min in fragmentation buffer (Ambion).
20 50 μg Poly(A)+ RNA was selected on oligo-dT25 DynaBeads (Invitrogen) per manufacturer's instructions, and fragmented at 95°C in fragmentation buffer (2 mM EDTA, 100 mM NaCO3/NaHCO3, pH 9.2) to a mean size of roughly 100 nt.
The 3' termini were end-labeled by addition of a biotinylated oligonucleotide (Affymetrix 7.5 mM Labeling Reagent) using terminal deoxynucleotidyl transferase (TdT) (Promega, Madison, WI, USA) as per a modified Affymetrix Labeling of Fragmented Double-Stranded DNA instructions [ 33].
Biotinylated cRNA was fragmented by heating with magnesium (as per the Affymetrix instructions) and this fragmented cRNA was hybridized to Mouse 430v2 GeneChips (Affymetrix, Santa Clara, Calif .. Data were analyzed using Expressionist Genedata AGG).
Amplified and biotin-labeled RNA was fragmented according to manufacturer's instructions.
Five micrograms of genomic DNA was fragmented and labeled according to instructions for labeling mRNA for antisense prokaryotic arrays (Affymetrix Inc .. Reactions containing 1 μg labeled DNA were used for hybridization at the Utah State University Center for Integrated Biosystems Affymetrix core facility (Logan, UT).
Briefly, polyadenylated RNA was purified using magnetic beads and fragmented according to the manufacturer's instructions.
5 μg of total RNA was labeled and fragmented according to the manufacturer's instructions (Affymetrix, Santa Clara, CA).
